DIAGNOSIS OF ALZHEIMER'S DISEASE

Abstract: An elevated level of serum amyloid P component (SAP) in a sample of cerebrospinal fluid from a subject relative to the level of SAP found in samples of cerebrospinal fluid from healthy individuals and/or individuals with a neurological disease other than Alzheimer's disease is indicative of Alzheimer's disease. Measurement of levels of SAP in cerebrospinal fluid enables diagnosis of Alzheimer's disease and/or monitoring of the progress of the disease.

Description: DIAGNOSIS OF ALZHEIMER'S DISEASE The present invention relates to the detection of human serum amyloid P component and the diagnosis of Alzheimer's disease. Amyloidosis is a disorder of protein metabolism in which soluble autologous proteins are deposited as insoluble fibrils, the accumulation and persistence of which causes disruption of structure and function of the affected tissues and leads to serious and usually fatal disease. There are many different forms of amyloidosis, each associated with a different fibril protein. Although microscopic amyloid deposition is always present in the elderly and rarely causes clinical problems, more substantial amyloidosis, especially in the vital organs, is associated with progressive, untreatable and usually fatal disease. The most common disorders associated with amyloidosis are Alzheimer's disease and maturity onset diabetes mellitus. Other forms of acquired and hereditary amyloidosis are rarer but cause serious morbidity and are generally fatal. Serum amyloid P component (SAP), a normal human plasma protein, is a universal constituent of amyloid deposits in all forms of amyloidosis as a result of its specific calcium-dependent binding affinity for amyloid fibrils (Pepys et al. Clin. Exp. Immunol. 1979, • 18.:284-293) . Cerebral amyloid is considered to be an integral part of the neuropathology of Alzheimer's disease, which disease is the most frequent cause of senile dementia and the fourth most common cause of death in the Western world. Clinical diagnosis of Alzheimer's disease is generally made according to the NINCDS-ADRDA criteria (McKhann et al. Neurology 1984;__4.:939-944) . However, diagnosis even according to those criteria is difficult and inaccurate, with significant numbers of false negatives and false positives (Cummings Bull Clin. Neurosci. 1991;___5:5-16) . Diagnosis even according to the above clinical criteria has been reported as accurate in as few as 50% of cases. A definitive diagnosis is rarely obtained during life. Although amyloid can be diagnosed by histological examination of the affected tissue, brain histology is seldom available during life and is usually only obtained if an alternative treatable condition is strongly suspected. Otherwise, histology to confirm clinical diagnosis of Alzheimer's disease is carried out only at autopsy. The present inventors have developed an in vivo method for the quantitative scintigraphy of visceral amyloid deposits, involving the use of radioisotope-labelled SAP, for example, 123 I- and "mTc-labelled SAP. Although this technique provides excellent images of amyloid deposits, the requirement for expensive, specialised equipment limits its use. It cannot be used for in vivo diagnosis of Alzheimer's disease. Furthermore, at present there is no in vitro method that can be used to provide an reliable diagnosis of Alzheimer's disease. SAP has been determined in serum by various different types of immunoassay and in various disease conditions, for example, in amyloidosis, connective tissue disease, infection and malignancy. Serum SAP levels have been compared in healthy controls and in patients with amyloidosis, but no differences in levels were found. Metabolic studies have shown major alterations in the turnover of SAP in patients with systemic amyloidosis although the steady state plasma concentration of the protein remains normal even during periods of massive deposition of SAP in myeloid. The present invention is based on our observation that individuals having Alzheimer's disease, for example as determined by the NINCDS-ADRDA criteria, have elevated levels of SAP in their cerebrospinal fluid in comparison with healthy subjects and with individuals with other neurological disease. In contrast, the levels of other plasma proteins, for example, serum albumin, IgG and α 2 -macroglobulin are substantially the same in the cerebrospinal fluid of all individuals tested. The present invention provides a method for the quantitative determination of serum amyloid P component in a sample of body fluid, characterised in the body fluid is cerebrospinal fluid. The present invention also provides a method of diagnosis of Alzheimer's disease, which comprises determining the level of serum amyloid P component in a sample of cerebrospinal fluid obtained from an individual relative to the level of serum amyloid P component determined in samples of cerebrospinal fluid obtained from healthy individuals and/or from individuals with other neurological diseases. The present invention also provides a method of testing for Alzheimer's disease in a subject, which comprises determining the level of serum amyloid P component in a sample of cerebrospinal fluid obtained from the subject and comparing the level of serum amyloid P component thus obtained with the level of serum amyloid P component determined in a sample of cerebrospinal fluid obtained from a healthy individual and/or from an individual with a neurological disease other than Alzheimer's disease thereby enabling diagnosis of Alzheimer's disease and/or monitoring of its progress. An elevated level of SAP in the sample under investigation relative to the levels in samples obtained from healthy individuals and/or from individuals with other neurological diseases is diagnostic of Alzheimer's disease. The method for the determination of serum amyloid P component may be any method capable of measuring the levels of the protein in cerebrospinal fluid, for example, measuring levels as low as 1 ng/ml. Preferably, the assay method comprises contacting a sample of cerebrospinal fluid with a specific binding partner for serum amyloid P component under condi¬ tions such that binding can occur between any serum amyloid P component present and the specific binding partner therefor, and determining any serum amyloid P component-specific binding partner complex formed. This method is itself part of the present invention. Accordingly, the present invention preferably provides a method of diagnosis of Alzheimer's disease, which comprises contacting a sample of cerebrospinal fluid with a specific binding partner for serum amyloid P component under condi¬ tions such that binding can occur between any serum amyloid P component present and the specific binding partner therefor, and determining the level serum amyloid P component-specific binding partner complex formed and hence the level of serum amyloid P component, relative to levels of SAP determined in the cerebrospinal fluid of healthy individuals and/or individuals with other neurological diseases. The present invention also provides a method of testing for Alzheimer's disease in a subject, which comprises contacting a sample of cerebrospinal fluid obtained from the subject with a specific binding partner for serum amyloid P component under conditions such that binding can occur between any serum amyloid P component present and the specific binding partner therefor, and determining the level of serum amyloid P component-specific binding partner complex formed, and comparing the level of complex formed with that formed in a sample of cerebrospinal fluid obtained from a healthy individual and/or from an individual with a neurological disease other than Alzheimer's disease and assayed under the same conditions, thereby enabling diagnosis of Alzheimer's disease and/or monitoring of its progress. An elevated level of SAP, as determined by the level of SAP- specific binding partner complex formed in a sample under investigation relative to the control levels, is diagnostic of Alzheimer's disease. The measurement of SAP levels in a series of samples of cerebrospinal fluid from an individual may be used to follow the progress of Alzheimer's disease in that individual and may be of considerable prognostic value. Measurement of SAP levels in CSF in different individuals may be carried out to promote the understanding of the natural progress of the disease and to evaluate the response of the disease to specific treatments. The specific binding partner for serum amyloid P component may be, for example, any one of a series of ligands capable of binding to SAP, for example, ligands to which SAP binds in the presence of calcium. Such ligands are described in the literature, and include DNA, chromatin, fibronectin, C4- binding protein and glycosaminoglycans, for example, 4,6- cyclic pyruvate acetal of β-D-galactose. A particularly useful example of a ligand for use in a quantitative assay for SAP is phosphoethanolamine, which can be immobilised on solid supports, for example, on Sepharose beads. Generally, however, a specific-binding partner is an antibody, either a monoclonal antibody or a polyclonal antibody. Anti-SAP monoclonal and polyclonal antibodies may be produced according to the usual methods, and a variety of such antisera have been described in the literature, for example, by Pepys et al. Clin. Exp. Immunol. 1978;32:119-124. When the specific binding partner in an assay is an antibody, the SAP is an antigen and the assay is an immunoassay. When a non-antibody specific binding partner for SAP, for example, a ligand is used, the assay is the direct equivalent of an immunoassay, and all references to immunoassays in the following description apply mutatis mutandis to corresponding assays using specific binding partners for SAP other than antibodies. An assay of the invention involving SAP and a specific binding partner therefor may be carried out by any immunoassay method. Numerous books and reviews articles describe the theory and practice of immunoassayε. Advice is given on the design of immunoassayε, for example, on the choice of homogeneous or capture format, on the characteristics and choice of solid substrate in the case of capture assays, on the nature and choice of label (signal generating system) , and on various other practical matters such as the composition of diluents and waεhing solutions. One example of a standard textbook is "ELISA and Other Solid Phase Immunoassays, Theoretical and Practical Aspects", Editors D.M. Kemeny & S.J. Challacombe, published by John Wiley, 1988. A further example is "practice and theory of enzyme immunoassays" by P. Tijssen in the series "laboratory techniques in biochemistry and molecular biology", general editors R.H. Burdon & P.H. van Knippenberg, Elsevier. There are essentially two types of immunoassay format, one in which an antibody or antigen of interest is captured selectively onto a solid phase from a sample under investigation, and another, a homogeneous phaεe aεsay format, in which the antibody or antigen of interest is detected in solution. It is particularly convenient to carry out either homogeneous or capture assays in small tubes or, especially, in the wells of microtitre plates (microwells) or using microparticles or beads because of the semi-automated and automated systems available for handling assays using εuch formats. In general, homogeneous assays are competitive assays, in which there are provided two labelled reagents, one comprising the antibody used for detection and the other comprising a labelled sample of the antigen under investigation i.e. SAP so that, when the antibody and antigen interact in solution, the two labels also interact, for example, to allow non-radiative transfer of energy captured by one label to the other label, with appropriate detection of the excited second label or quenched first label, for example, by fluori etry, magnetic resonance or enzyme measurement. Addition of the sample under investigation results in modification of the interaction of the labelled antibody and labelled antigen and so to a different level of εignal. In many cases, the signal is preferably a colour change. In an immunoasεay in which an antigen i.e. SAP iε captured from the εample under investigation and immobilised within an immunoasεay veεεel, the solid phase on which the capture occurs is, for example, beads of a polymeric substance, especially a synthetic plastics material; particles, for example, εo-called "latex" particles, stabiliεed blood, bacterial or fungal cells, spores, gold or other metallic sols, and proteinaceous colloids. The size of the particles is generally from about 0.1 to 5 microns, and beads generally larger, for example, from about 2 mm to 10 mm. An antigen is generally captured on the solid support by means of a specific antibody that has been coated on the solid support. In a solid phase assay, the captured antigen i.e. SAP is generally detected by any means that will give a signal, for example, by the use of a labelled molecule or particle that will react with the captured antigen, generally a labelled antibody. The detectable signal for a homogenouε or capture assay may be optical, radio-active or physico-chemical, and may be provided directly by labelling a molecule or particle as described above, for example, with a dye, radiolabel, electroactive species, magnetically resonant species or fluorophore; or indirectly by labelling a molecule or particle as described above with an enzyme itself capable of giving rise to a measurable change of any εort. Alternatively, for a capture assay, the signal may result from agglutination, a diffraction effect or a birefringement effect occurring if any of the εolid phase comprises appropriately sized particles, for example, aε described above. In general ELISAs are preferred although radioimmunoasεayε may find particular applicationε. Immunoassays described previously for the determination of SAP in serum or plaεma include: electroimmunoassay (Pepys et al. 1978, loc. cit.), radioimmunoassay (Skinner et al. J. Lab. Clin. Med. 1979;9_4:633-638) and ELISA (Serban et al. J. Immunol. Methods 1986;90:159-164, Bannikova et al. B. Exp. B. Med. 1989;108.:1022-1025, and Saile et al. Clin. Chem. 1991;_Z:1742-1745). Any of the above methods and any other quantitative immunoassay (including specific binding asεayε uεing, for example, immobilised phosphoethanolamine) may be used for the determination of SAP in samples of cerebrospinal fluid according to the present invention. Aε indicated above, the solid support for a capture asεay iε, for example, plastics beads, microparticles or the wells or cups of microtitre plates. Automated systems are available for handling such assay formats. Other solid supports that may be used include membranes, sheets and stripε, for example, of a porouε, fibrous or bibulous material, for example, of nylon, nitrocelluloεe, polyvinylidene or paper, for example, diazotized paper. Porouε, fibrouε or bibulouε membraneε, sheets and strips may be incorporated into assay devices. Assay devices generally incorporate, within a rigid support or a housing, all the reagents required for carrying out an assay. The sample is generally applied to the device at a predetermined sample application zone, for example, by pouring or dripping the sample on the zone, or by dipping the relevant part of the device into the sample. The arrangement of the reagentε within the device and, in some cases, the mechanical interactions between different partε of the device, which mechanical interactions may occur automatically during the operation of the device or may be brought about by the user of the device, is such that, on application of the sample to the device, the various reagentε are brought into contact with one another in the correct sequence for the immunoassay to be carried out. Assay devices have the particular advantage that they can be used out without the need for laboratory facilities, for example, in a doctor's surgery. The following Example illustrates the present invention. EXAMPLE Cerebrospinal fluid (CSF) was obtained by lumbar puncture from 51 patients with probable Alzheimer's disease as diagnosed in accordance with the clinical NINCDS/ADRDA criteria, mean age (SD) , 70 (8) years, and from 17 healthy normal subjects, mean age (SD), 66 (11) years. Samples were also obtained from 7 patients with functional psychiatric disorders, mean age (SD), 62 (12) years, and 26 patients with structural diseases of the central nervous system, including multi-infarct dementia (n=3), Parkinson's disease (n=3), Pick's disease (n=3), multiple εcleroεis, intracerebral tumours and anoxic damage (2 caseε each) and other chronic neurological diεorderε, mean age (SD), 68 (9) yearε. There were no significant differences in age or sex within the four different groups or between the Alzheimer'ε diεeaεe group and the remaining 50 non-Alzheimer's disease subjectε collectively. Additional εerum samples, obtained at the same time aε lumbar puncture, were available in 3 normal healthy εubjectε and 8 patients with Alzheimer's disease. The CSF concentrations of albumin, IgG, SAP and α^-macro- globulin, molecular weights 66, 150, 254 and 720 kD respectively, were assayed aε followε: Albumin: "Rocket" immunoelectrophoreεiε uεing Dako A001 antibodieε and calibrated with LCV Behring standard serum, range 36-572 μg/ml. IgG: Single radial immunodiffusion using Dako A424 antibodies and calibrated with LCV Behring standard serum, range 5-80 μg/ml. c.2~~ macroqlobul n: Immunoradiometric assay uεing Dako A033 antibodieε immobilised on CNBr-Sepharose beadε for antigen capture, and radiolabelled with 1 25I for assay; purified α - macroglobulin kindly provided by Dr. Alan Barnett (Strangeways Institute), was used for calibration and the range was 1-20 μg/ml. SAP: Immunoradiometric asεay comprising a polyclonal IgG fraction of sheep anti-SAP serum coated on "Dynatech" flexible PVD icrotitre plates for antigen capture, and 1 25I- labelled affinity purified SAP antibodies for aεεay; iεolated pure SAP and native SAP in whole normal human εerum were uεed to calibrate the assay, which had a range of 0.1-1,000 mg/ml. Duplicate CSF samples were asεayed and the inter- and intra- assay coefficients of variation were less than 10% in each case. The CSF concentrations of albumin, IgG and α -macroglobulin in all individuals (see following Table) were comparable to those in other published studies and there were no significant differences between patientε with Alzheimer's disease and control subjects. The CSF concentration of SAP in healthy normal subjects and all other patients without Alzheimer's disease was mean (SD), 8.5 (9.1) ng/ml, with a range of 1.0-44.0 ng/ml. Amongεt patientε with Alzheimer's disease (n=51) the CSF concentrations of the SAP was significantly elevated, mean (SD), 12.8 (11.9) ng/ml, range 1.0-40.0 ng/ml. The plasma concentration of SAP in 3 healthy subjects and 8 patientε with Alzheimer'ε disease was mean ( S D), 31 ( 11 ) mg/ml, not significantly different from that in the normal population. TABLE CSF concentration Patients with AD Individuals without AD (n = 51) (n = 50) Albumin (μg/ml) 260 224 n.s. IgG (μg/ml) 32 31 n.s. SAP (ng/ml) 12.8 8.5 P <0.03* cx_-macroglobulin (μg/ml) 1.7 1.7 n.s. P = 0.029, non-paired t-test; 0.0125, Mann Whitney U-test. Results for other proteins did not reach significance P >0.05 (n.s.).

Claims: CLAIMS : 1. A method of testing for Alzheimer's disease in a subject, which comprises determining the level of serum amyloid P component in a sample of cerebrospinal fluid obtained from the subject and comparing the level of serum amyloid P component thus obtained with the level of serum amyloid P component determined in a sample of cerebrospinal fluid obtained from a healthy individual and/or from an individual with a neurological diεeaεe other than Alzheimer'ε diεeaεe thereby enabling diagnosis of Alzheimer's disease and/or monitoring of its progress. 2. A method as claimed in claim 1, wherein serum amyloid P component iε determined by contacting a sample of cerebrospinal fluid with a specific binding partner for εerum amyloid P component under conditions such that binding can occur between any serum amyloid P component present and the specific binding partner therefor, and determining the level of serum amyloid P component-specific binding partner complex formed. 3. A method as claimed in claim 2, wherein the specific binding partner is a ligand to which serum amyloid P component binds in the presence of calcium. 4. A method as claimed in claim 3, wherein the ligand iε phosphoethanola ine. 5. A method as claimed in claim 2, wherein the specific binding partner iε a monoclonal or polyclonal antibody. 6. A method as claimed in claim 5, wherein the serum amyloid P component determination iε carried out as an enzyme-linked immunosorbent assay (ELISA).

Inventors: HAWKINS PHILIP NIGEL, PEPYS MARK BRIAN, HAWKINS, PHILIP, NIGEL, PEPYS, MARK, BRIAN
Applicants: HAWKINS PHILIP NIGEL, PEPYS MARK BRIAN, ROYAL POSTGRAD MED SCHOOL, HAWKINS, PHILIP, NIGEL, PEPYS, MARK, BRIAN, ROYAL POSTGRADUATE MEDICAL SCHOOL
Application year: 1994
Main classification: G01N33
IPC codes: G01N33